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Browsing by Author "Song, Fengyu"
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Item The Axolotl Fibula as a Model for the Induction of Regeneration across Large Segment Defects in Long Bones of the Extremities(Public Library of Science, 2015) Chen, Xiaoping; Song, Fengyu; Jhamb, Deepali; Li, Jiliang; Bottino, Marco C.; Palakal, Mathew J.; Stocum, David L.; Department of Biology, School of ScienceWe tested the ability of the axolotl (Ambystoma mexicanum) fibula to regenerate across segment defects of different size in the absence of intervention or after implant of a unique 8-braid pig small intestine submucosa (SIS) scaffold, with or without incorporated growth factor combinations or tissue protein extract. Fractures and defects of 10% and 20% of the total limb length regenerated well without any intervention, but 40% and 50% defects failed to regenerate after either simple removal of bone or implanting SIS scaffold alone. By contrast, scaffold soaked in the growth factor combination BMP-4/HGF or in protein extract of intact limb tissue promoted partial or extensive induction of cartilage and bone across 50% segment defects in 30%-33% of cases. These results show that BMP-4/HGF and intact tissue protein extract can promote the events required to induce cartilage and bone formation across a segment defect larger than critical size and that the long bones of axolotl limbs are an inexpensive model to screen soluble factors and natural and synthetic scaffolds for their efficacy in stimulating this process.Item BENS, a novel regulator of bone/cartilage healing(2013) Labban, Nawaf Yousef; Windsor, L. Jack; Song, Fengyu; Ghoneima, Ahmed; Bruzzaniti, Angela; Allen, Matthew R.; Cameron, Jo AnnEnhancing osteoblast proliferation, survival, and extracellular matrix protein secretion are potential therapeutic approaches to treat bone fractures and diseases such as osteoporosis. BENS is a traditional medicine used in many countries such as India for thousands of years to treat many diseases including bone diseases. In this study, molecular, cell-based and in vivo approaches were utilized to investigate the effects of BENS on bone and cartilage regeneration. An osteosarcoma cell line (MG63) was incubated in serum free media with and without 0.8 mg/ml of BENS. BENS significantly increased cell survival up to 30 days and these cells retained their ability to proliferate in fresh media with serum. After adding BENS, there were statistically significant decreases in the expression of both anti-apoptotic and pro-apoptotic proteins. An in vivo non-critical size segmental bone defect Xenopus system was used to evaluate the ability of BENS to enhance cartilage formation. After a small segment of the anterior hemisection of the tarsus bone was excised, the frogs were divided into three groups and given subcutaneous injections of either phosphate-buffered saline or BENS once daily for 30 days and then bone/cartilage formation evaluated. The total cartilage area/total section area was significantly increased (2.6 fold) in the BENS treated samples. In an osteoporotic rat model, the anabolic properties of BENS on bone mass were assessed by histomorphometric analyses. Ovariectomized (OVX) rats received daily intraperitoneal injections for 4 weeks. Bone formation rates (BFRs) for the cortical periosteal bone surface of the midshaft tibia were 383.2, 223.9, 308.8, 304.9, and 370.9 µm3/µm2/year, and for the trabecular surface were 82.2, 113, 212.1, 157, and 165 µm3/µm2/year for the sham, OVX, PTH, 3 mg/kg BENS, and 30 mg/kg BENS groups, respectively. BENS increased both trabecular and cortical BFRs. It generated better results on cortical periosteal bone surface than did PTH. Taken together, these findings suggest that BENS promotes osteoblast survival due to its effects on altering the balance between pro-apoptotic and anti-apoptotic proteins. In addition, in vivo studies revealed that BENS enhanced cartilage formation in Xenopus and BFRs in rats. Therefore, BENS may possess anabolic bone/cartilage properties.Item Effect of nicotine on streptococcus mutans(2014-11) Huang, Ruijie; Gregory, Richard L.; Tu, Wanzhu; Windsor, L. Jack; Wu, Christine; Song, FengyuStreptococcus mutans is a key contributor to dental caries. Smokers have increased caries, but the association between tobacco, nicotine, caries and S. mutans growth is little investigated. In the first section, seven S. mutans strains were used for screening. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm inhibitory concentration (MBIC) were 16 mg/ml (0.1 M), 32 mg/ml (0.2 M), and 16 mg/ml (0.1 M), respectively, for most of the S. mutans strains. Growth of planktonic S. mutans cells was significantly repressed by 2.0-8.0 mg/ml nicotine concentrations. Biofilm formation and metabolic activity of S. mutans was increased in a nicotine-dependent manner up to 16.0 mg/ml. Scanning electron microscopy (SEM) revealed higher nicotine-treated S. mutans had thicker biofilm and more spherical bacterial cells than lower concentrations of nicotine. In the second section, confocal laser scanning microscopy (CLSM) results demonstrated that both biofilm bacterial cell numbers and extracellular polysaccharide (EPS) synthesis were increased by nicotine. Glucosyltransferase (Gtf) and glucan binding protein A (GbpA) protein expression of S. mutans planktonic cells were upregulated, while GbpB protein expression of biofilm cells were downregulated by nicotine. The mRNA expression of those genes were mostly consistent with their protein results. Nicotine was not directly involved in S. mutans LDH activity. However, since it increased the total number of bacterial cells in biofilm; total LDH activity of S. mutans biofilm was increased. In the third section, a PCR-based multiple species cell counting (PCR-MSCC) method was designed to investigate the effect of nicotine on S. mutans in a ten mixed species culture. The absolute S. mutans number in mixed biofilm culture was increased but the percentage of S. mutans in the total number of bacterial cells was not changed. In conclusion, nicotine enhanced biofilm formation and biofilm metabolism of S. mutans, through stimulating S. mutans planktonic cell Gtfs and Gbps expression. This leads to more planktonic cells attaching to dental biofilm. Increased S. mutans cell numbers, in biofilms of single species or ten mixed species, resulted in higher overall LDH activity. More lactic acid may be generated and contribute to caries development in smokers.Item Effects of DynaMatrix® Membrane on Angiogenic Cytokine Expression From Human Dental Pulp Stem Cells(2013) Baker, Ryan William; Spolnik, Kenneth Jacob, 1950-; Ehrlich, Ygal; Vail, Mychel Macapagal, 1969-; Song, Fengyu; Legan, Joseph J.; Zunt, Susan L., 1951-; Windsor, L. JackThe aim of this current study was to determine if the exposure of human dental pulp stem cells (HDPSC) to the DynaMatrix membrane will result in an increased production of angiogenic cytokines that are critical for pulp/root regeneration. Angiogenesis cytokine arrays have been established as a viable method for assessing expression of cytokines.20 HDPSC were chosen as they are expected to be found in the apical papilla and the infected immature root canal system of teeth that current regenerative endodontic techniques are designed to treat.Item Effects of tobacco on human gingival fibroblasts(2011) Zhang, Weiping; Windsor, L. Jack; Song, Fengyu; Kowolik, Michael J.; Lee, Chao-Hung; Subramaniam, Denise RogersThe negative heath consequences of smoking are widely recognized, but there are still about 20% of the people in United States using tobacco products. Cigarette smoke condensate (CSC), the particulate matter of cigarette smoke, is comprised of thousands of chemicals (e.g., nicotine). Secondary only to bacterial plaque, cigarette smoking is a major risk factor for periodontal disease. Human gingival fibroblasts (HGFs) are the main cellular component of periodontal connective tissues. During the development of periodontal disease, collagen degradation occurs. Collagen is the major extracellular matrix component of the gingiva. The major extracellular matrix degrading enzymes produced by the HGFs are the matrix metalloproteinases (MMPs). The MMPs are mainly modulated by the tissue inhibitors of metalloproteinases (TIMPs). In this dissertation, three studies aimed at understanding the effects of tobacco on human gingival fibroblasts and their mechanisms have been conducted: the effects of CSC on HGF-mediated collagen degradation; comparison of the effects of CSC on HGFs with that of nicotine; and the combined effects of CSC and bacteria on HGFs. The cell proliferation of HGFs decreased and cytotoxicity increased in HGFs treated with increasing concentrations of CSC. CSC increased the collagen degrading ability of the HGFs by altering the production and localization of MMPs and TIMPs. Nicotine is one of the major components and the most pharmacologically active agent in tobacco. The percentage of nicotine in the CSC was 2.4%. CSC (100 µg/ml) increased the collagen degrading ability of the HGFs by affecting membrane associated MMP-2, MMP-14, and TIMP-2, but the level of nicotine in the CSC may only play a limited role in this process. Porphyromonas gingivalis (P. gingivalis) is an opportunistic pathogen involved in periodontal disease. The combined effects of CSC and P. gingivalis supernatant increased HGF-mediated collagen degradation by destroying the balance between the MMPs and TIMPs at the protein and mRNA levels. This project demonstrated that tobacco (with or without P. gingivalis) increased HGF mediated collagen degradation, as seen in the periodontal disease, through altering the MMPs and TIMPs.Item INTERACTIONS OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS WITH TOBACCO TREATED STREPTOCOCCUS MUTANS(Office of the Vice Chancellor for Research, 2013-04-05) Gupta, Vinayak; Windsor, L. Jack; Song, Fengyu; Gregory, Richard L.Streptococcus mutans (S. mutans) is a major contributor to dental caries. Previous research has shown that there is a positive relationship between smoking and dental carries, however the pathway of S. mutans growth is not yet understood. Tobacco use affects the cardiovascular system and increases the rate of cardiovascular disease among smokers. However, the effects of tobacco on the endothelial cells that line blood vessels are not yet fully understood. Thus, the objective of this study was to examine some of the effects that a periodontal pathogen such as S. mutans treated with cigarette smoke condensate (CSC) and nicotine have on human umbilical vein endothelial cells (HUVEC’s). The S. mutans was grown at 37°C and then the planktonic cells were harvested, washed with saline, and then killed with formaldehyde. To standardize the samples, they were diluted to the same OD at 600nm wavelength using a spectroscope. The HUVEC were cultured in Endothelial Basal Medium-2 and plated in 12 well plates and exposed to the P. gingivalis cells and supernatants and after 72 hours, lactate dehydrogenase (LDH) assays will be used to cytotoxicity. Non-toxic amounts of the cells and supernatants will then be used to treat HUVEC cells for 72 hours before the media is collected and analyzed for cytokine/growth factor expression by protein arrays. Second messenger signaling pathways will be analyzed with ERK and JNK antagonists and agonists to determine the pathway of up regulation of S. mutans. A better understanding of the detrimental effects that tobacco has on the underlining causes of periodontal disease can advance the quest of controlling the disease.Item Preparation and Evaluation of Antibacterial Dental Glass-ionomer Cements(2010-10-22) Guo, Xia; Xie, Dong; Na, Sungsoo; Song, FengyuThe functional quaternary ammonium salts (QAS) and their constructed polyQAS or PQAS were synthesized, characterized and formulated into a novel antibacterial glass-ionomer cement. Compressive strength (CS) and Streptococcus mutans (S. mutans) viability were used to evaluate the mechanical strength and antibacterial activity of the cements. Fuji II LC cement was used as control. The specimens were conditioned in distilled water at 37 oC for 24 h prior to testing. The effects of the substitute chain length, loading as well as grafting ratio of the QAS and aging on CS and S. mutans viability were investigated. Chapter 2 describes how we studied and evaluated the formulated antibacterial glass-ionomer cement by incorporating QAS chloride-containing polymer into the formulation. The results show that with PQAS addition, the studied cements showed a reduction in CS with 25-95% for Fuji II LC and 13-78% for the experimental cement and a reduction in S. mutans viability with 40-79% for Fuji II LC and 40-91% for the experimental cement. The experimental cement showed less CS reduction and higher antibacterial activity as compared to Fuji II LC. The long-term aging study indicates that the cements are permanently antibacterial with no PQAS leaching. Chapter 3 describes how we studied and evaluated the formulated antibacterial cements by changing chain length, type of halide, loading, grafting ratio and aging time. The results show that the effects of the chain length, loading and grafting ratio of the QAS were significant. Increasing chain length, loading, grafting ratio significantly enhanced antibacterial activity but reduced CS. The experimental cement showed less CS reduction and higher antibacterial activity as compared to Fuji II LC. The long-term aging study indicates that the cements are permanently antibacterial with no PQAS leaching. There was no significant difference between QAS bromide and QAS chloride, suggesting that we can use QAS bromide directly without converting bromide to chloride. In summary, we have developed a novel PQAS-containing antibacterial glass-ionomer cement. The cement has demonstrated significant antibacterial activities. Our experimental cement is a promising system because the reduced strength of the cement with addition of PQAS is still above those demonstrated by original commercial cement Fuji II LC without any PQAS addition. It appears that the experimental cement is a clinically attractive dental restorative that can be potentially used for long-lasting restorations due to its high mechanical strength and permanent antibacterial function.Item The Responses of Human Neutrophils to Tobacco Smoke Components(2012) Al-Shibani, Nouf Khider; Windsor, L. Jack; Song, Fengyu; Kowolik, Michael J.; Goodpaster, John V. (John Vincent); Subramaniam, Danise RogersTobacco smoking is considered a major modifiable risk factor for periodontal disease. Tobacco contains about 6700 compounds and almost 4000 compounds of these have been identified in tobacco smoke. Nicotine is the addictive ingredient in tobacco and has been shown to affect multiple cellular processes. Cigarette smoke condensate (CSC) is the particulate matter of smoke. It is believed to be a powerful inducer of inflammatory responses. Neutrophils are the first line of host defense and are critical cells in the maintenance of periodontal health through their role in the control of bacteria, but they can also contribute to the progression of periodontal disease by the production and release of reactive oxygen species (ROS). Virulence factors from periodontal pathogens, such as Porphyromonas gingivalis (P. gingivalis), stimulate the respiratory burst of neutrophils. In this dissertation, three studies aimed at understanding the oxidative activity of neutrophils when stimulated with either nicotine, cigarette smoke condensate (CSC) or four other components of tobacco smoke (2-naphthylamine, hydroquinone, acrolein, and acetaldehyde) with or without P. gingivalis supernatant. The release of matrix metalloproteinase-9 (MMP-9) was also examined. ROS production increased significantly when the neutrophils were stimulated with nicotine. P. gingivalis induced the maximum ROS production when compared to all the other components examined. The combination of nicotine and P. gingivalis did not have an additive effect on ROS production. Nicotine significantly increased the MMP-9 release from the neutrophils. On the contrary, CSC inhibited ROS production at all the concentrations examined. The combination of CSC and P. gingivalis resulted in the inhibition of ROS production. MMP-9 release was also increased from the CSC-treated neutrophils. The four other tobacco smoke components examined affected ROS production and MMP-9 release differently. These projects demonstrated that CSC inhibited the ROS production from neutrophils, which can be attributed to several components in tobacco smoke that may include acrolein and hydroquinone. More research is needed to determine the mechanisms of inhibition and if other tobacco components are involved in ROS inhibitionItem Study of Fulvic Acid: A Natural Dietary Supplement(Office of the Vice Chancellor for Research, 2014-04-11) Syed, Ghayasul I.; Gregory, Richard L.; Windsor, L. Jack; Song, FengyuShilajit is a substance found in parts of Asia. Although there have been no clinical studies, it is used by the locals and is marketed because it is thought to have antiseptic, anti-inflammatory and pain suppressing effects. Fulvic acid (F-A) is a major constituent of shilajit and was used in the analysis of the anti-pathogenic tendencies of shilajit and cytotoxic effects on human cells of the oral cavity. The bacterial study was performed on Streptococcus mutans, a normal flora of the oral cavity. The idea was to test the metabolic activity of the bacteria in F-A-containing media. Menadione-XTT reagent was used for this. The bacterial biofilm was allowed to grow in TSBS in a microtiter plate of 96 wells. The F-A solution of different concentration were introduced into each well in a gradually decreasing amount and the last control wells had a zero concentration. The XTT reagent was introduced and after incubation the biofilm of S. mutans reduced the XTT to an orange color, the change in color was detected by measuring the absorbance at 490nm. Between 2.5% to 5.0% of F-A the wells showed signs of decreased activity. The numbers indicated that absorbance of the wells with concentrated F-A was lower compared to the wells with more diluted F-A solutions. From this it can be concluded that F-A had a negative effect on the growth and metabolic activity of S. mutans. For human testing, pulp and fibroblast cells were subjected to different concentrations of F-A. The cytotoxicity was measured by the amount of Lactate Dehydrogenase released from the treated cells (sign of damage). Overall, the experiment validates the potency of F.A as an effective antibacterial. Further testing is needed but the compound shows promise and can be employed as an effective ingredient of mouthwash and other such antiseptic products.